During the past decades the interest for preparation and commercialisation of human milk oligosaccharides (HMOs) has been increasing steadily. The importance of HMOs is directly linked to their unique biological activities such as antibacterial, antiviral, immune system and cognitive development enhancing activities.
The tetrasaccharide Galpβ1-4GlcNAcpβ1-3Galpβ1-4Glc (lacto-N-neotetraose, LNnT, Scheme 1) is one of the oligosaccharides occurring in human milk [Kuhn et al. Chem. Ber. 1962, 95, 513 and 518, Kobata Methods Enzymol. 1972, 28, 262]. LNnT act as bacterial receptor for pneumococci and it was found to be useful in the recognition of the acceptor specificity of glycosyltransferases, the substrate specificity of glycosidases and the structure of antigenic determinants. Furthermore LNnT represents a core structural element of more complex oligosaccharides, in glycolipids and in glycoproteins (paragloboside, 6-sialyl-LNnT, etc.) with various physiological activities.

To date, access to large volumes of LNnT has not been possible by using isolation, biotechnology and synthetic methodologies. The isolation of LNnT from human milk is rather difficult even in milligram quantities due to the presence of a large number of similar oligosaccharides.
Enzymatic synthesis of LNnT consists of incubation of lacto-N-triose II with UDP-galactose in the presence of a galactosyltransferase [Sabesan et al. J. Am. Chem. Soc. 1986, 108, 2068; EP-A-870841] or with lactose in the presence of β-D-galactosidase [Murata et al. Glycoconj. 1999, 16, 189; JP 10-234394 A]. Enzymatic galactosylation of the proper trisaccharide bound to solid support was also elaborated [Blixt et al. Carbohydr. Res. 1999, 319, 80; Renaudie et al. ibid. 2004, 339, 693]. These complex enzymatic systems represent very expensive methodologies and difficult purification protocols for scale-up productions of LNnT.
Total synthetic procedures towards LNnT published [Zurabyan et al. Soviet J. Bioorg. Chem. 1978, 4, 679; Paulsen et al. Carbohydr. Chem. 1987, 169, 105; Aly et al. ibid. 1999, 316, 121] comprise plenty of reaction steps, protecting group manipulations and chromatographic purification, poor yields, and provide only milligram quantity of LNnT, thus they do not offer attractive techniques for large scale preparation. Synthesis of 1-O-benzyl-LNnT as benzyl analogue of paragloboside has also been published [Ponpipom et al. Tetrahedron Lett. 1978, 20, 1717].
When isolated from natural source [Kuhn et al. Chem. Ber. 1962, 95, 513] or made by enzymatic way [EP-A-1405856] LNnT was characterized as a crystalline material.
With respect of LNnT and intermediates towards it there is still a need for crystalline products which may simplify isolation, purification and formulation problems so far envisaged.